monocytogenes strains without the need for additional genetic manipulations to introduce the nisRK genes into the chromosome of each strain. Plasmid pAKB, a derivative of plasmid pNZ8048 this website carrying the nisA promoter, was constructed for the planned overexpression experiments. To construct this plasmid, Belinostat molecular weight a cassette comprised of the nisRK genes cloned downstream of the L. monocytogenes hly promoter was introduced into pNZ8048 to ensure efficient expression of these genes in L. monocytogenes [15]. The lmo1438 gene was then cloned downstream

of the Pnis promoter in pAKB to produce plasmid pAKB-lmo1438. Before starting the experiments on overexpression of the lmo1438 gene, the susceptibility of L. monocytogenes to nisin was examined, since nisin is an inducer of the NICE system but it can affect or inhibit the growth of L. monocytogenes when used at high concentrations. The level of nisin required to completely inhibit the growth of L. monocytogenes EGD and of L. monocytogenes carrying the pAKB plasmid lacking an insert (used as a negative control in subsequent experiments) was over ten times higher than the concentration used previously Semaxanib purchase to induce

the NICE system in L. monocytogenes [15]. Furthermore, growth curves were plotted for L. monocytogenes pAKB grown in the presence of different concentrations of nisin as well as in the absence of this inducer to determine the concentration of nisin that has no effect on growth. These preliminary experiments showed that 15 μg/ml was the maximum concentration of nisin that did not cause any changes in the growth rate of the control strain. At higher nisin concentrations, including that used previously (45 Prostatic acid phosphatase μg/ml) to induce NICE in L. monocytogenes [15], a slight reduction in the growth rate of L. monocytogenes pAKB was observed during the exponential phase, compared to growth in the absence of nisin. The differences between the optimal

nisin concentrations for growth and induction determined here and those established by Cotter et al. [15] may be due to the differential susceptibility of the strains EGD and LO28 to this peptide. To confirm that nisin induced overexpression of the lmo1438 gene in L. monocytogenes pAKB-lmo1438, the cell membrane proteins of this strain and the control strain were analyzed. SDS-PAGE of isolated membrane proteins revealed the presence of an additional protein in L. monocytogenes pAKB-lmo1438 grown in the presence of 15 μg/ml nisin (Figure 1). The estimated mass of this additional protein was approximately 80 kDa, which corresponds to the predicted mass of Lmo1438 (79.9 kDa). The additional protein was detected at both 2 and 24 h following induction, but it was not observed when L. monocytogenes pAKB-lmo1438 was grown in the absence of nisin (data not shown). Figure 1 Overexpression of the lmo1438 gene in L. monocytogenes. Membrane proteins were isolated from L. monocytogenes pAKB (lane 1) and L.